新生小鼠巨细胞病毒肝炎模型的建立

目的 探讨建立鼠巨细胞病毒(MCMV)感染致新生小鼠肝炎模型的可行性.方法 48只日龄24 h内新生BALB/c小鼠随机分为实验组和对照组,实验组每只小鼠腹腔注射MCMVSmith株20μl TCID50(104.31/0.1 ml),对照组注射无菌生理盐水20μl,注射第3、7、14天取血清和肝脏,检测血清ALT水平,肝脏组织HE染色后用光镜检查组织病理损害,同时提取肝脏组织的DNA,应用MCMV及β-actin引物行PCR扩增,然后跑电泳和测序.结果 实验组小鼠ALT水平(U/L)在3天时即明显升高,7天达高峰,14天时有所下降,与对照组比较,差异均有统计学意义[3天:(58.7±11.5)比(25.0±10.6),7天:(169.6±57.4)比(25.1±8.4),14天:(157.3±15.5)比(26.5±9.4),P均＜0.01].实验组小鼠肝组织内均可见肝细胞气球样变性,点灶状坏死,门管区炎性细胞浸润,肝细胞核内病毒包涵体,7天时最严重;对照组肝细胞无相似病理变化.实验组MCMV-DNA PCR电泳全部出现阳性条带,阳性条带测序结果与MCMV基因序列的同源性完全相符.结论 MCMV能侵袭BALB/c新生小鼠引起肝炎,这种模拟人类巨细胞病毒肝炎的新生小鼠模型的建立为该病动物实验研究提供了可能.     

巨细胞病毒性疾病的诊断

小儿巨细胞病毒性疾病是由巨细胞病毒（CMV）引起的疾病,简称CMV病。在作诊断时,必须了解我国小儿主要由母婴传播获得感染,多在婴幼儿期患病。以后病毒长期潜伏在宿主体内。由于年龄特点、个体差异和各脏器致敏性不同,小儿CMV病临床变化众多。总的来说,新生儿（包括胎儿）可以产生以中枢神经系统受损为主的疾病;婴儿感染后多数可有不同程度的肝胆受损,少数发生间质性肺炎;其他大年龄小儿,仅为无症状性CMV感染（有时CMV也可复制,但不引起疾病）。诊断CMV性疾病必须具备活动性CMV感染和宿主脏器病变临床表现这两个基本条件。由下列检测中任何一项阳性即为活动性CMV感染,如病毒分离阳性,病毒抗原pp65检测阳性,病毒核酸检测特异性CMV mRNA阳性,血清学检测CMVIgM阳性。由于婴儿期CMV病以肝胆和肺部疾病多见,除非直接从病变组织中检出活动性CMV感染标记物,才可确定是由CMV引起,若从血、尿等标本查得,就必须排除能够引起同样临床表现的其他病因和病原。     

巨细胞病毒活动性感染的分子生物学特点

巨细胞病毒（ＣＭＶ）活动性感染可引起严重的疾病，在此期间，病毒大量复制，并伴随着不同程度的基因表达。主要即刻早期启动子（ＭＩＥＰ）与ＣＭＶ组织特异性表达高度相关，细胞分化可能诱发活动性感染，本文就活动性感染的部分分子生物学特性的变化及潜伏感染激活的主要发生机制作一综述。     

巨细胞病毒和巨细胞病毒感染的诊断

巨细胞病毒感染（ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓｉｎｆｅｃｔｉｏｎ）是由人巨细胞病毒（ｈｕｍａｎｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ,ＣＭＶ）引起［１］,在我国相当普遍,且大多在幼年时期发生。据我们１９９５年至１９９７年连续３年对１６７８例就诊小儿筛查结果发...     

巨细胞病毒活动性感染的分子生物学特点

巨细胞病毒（ＣＭＶ）活动性感染可引起严重的疾病，在此期间，病毒大量复制，并伴随着不同和蔼的基因表达。主要即刻早期启动子（ＭＩＥＰ）与ＣＭＶ组织特异性表达高度相关，细胞分化可能诱发活动性感染，本文就活动性感染的部分分子生物学特性的变化及伏感染激活的主要发生机制作一综述。     

用重组病毒株建立鼠巨细胞病毒性肝炎的实验模型

用插入LacZ基因的重组病毒株建立BALB/c小鼠巨细胞病毒 (murinecytomegalovirus ,mCMV)性肝炎模型。20只BALB/c甲基强的松龙免疫抑制小鼠 ,腹腔接种1×106PFU病毒悬液 ,动态观察血单个核细胞和组织中mCMV感染及肝组织病理变化。结果 :①mCMV感染小鼠血单个核细胞中X -gal染色阳性细胞在48h时开始出现 ,第3天有所减少 ,在第6天达到高峰;②肝、肺、肾组织病毒感染72h后可见 ,在第12天斑点数最多 ,以肝组织病毒感染多见;③肝组织病变在病毒感染后96h开始出现 ,在第12天达到高峰。提示 :利用重组mCMV建立的mCMV性肝炎小鼠模型 ,在感染后2周仍保持较高感染水平 ,为CMV的研究提供了一个良好的实验模型。     

呼吸道合胞病毒和人巨细胞病毒混合感染对感染细胞的影响

目的 探讨呼吸道合胞病毒（ＲＳＶ）和人巨细胞病毒（ＨＣＭＶ）混合感染对感染细胞的影响。方法 用四甲基偶氮唑盐（ＭＴＴ）染色方法检测下列四组标本的吸光度Ａ值（曾称光密度ＯＤ）,包括：未感染病毒细胞组、ＲＳＶ感染细胞组、ＲＳＶ和ＨＣＭＶ同时混合感染细胞组、ＨＣＭＶ感染１２ｈ后再感染ＲＳＶ细胞组（每组２０孔共８０孔）;流式细胞分析仪检测下列六组标本细胞分裂周期,包括：正常细胞组、ＲＳＶ感染组细胞、ＨＣＭ     

人巨细胞病毒糖蛋白B基因型与婴儿巨细胞病毒性肝炎的关系

人巨细胞病毒（HCMV）感染在我国广泛存在,肝脏是婴儿期HCMV感染最主要的靶器官之一。本文应用套式PCR（nPCR）加限制性长度多态性分析（RFLP）对HCMV主要包膜糖蛋白B（glycoprotein B,gB）基因进行分型,并分析2003年10月-2005年4月我院婴儿巨细胞病毒性肝炎与gB基因型的关系。     

Role of cysteinyl leukotrienes in airway inflammation and responsiveness following RSV infection in BALB/c mice

Cysteinyl leukotrienes (CysLTs) contribute to the development of airway obstruction and inflammation in asthma; however little information is available on the role of these molecules in the pathophysiology of respiratory syncytial virus (RSV) bronchiolitis. This study was designed to evaluate the effects of RSV infection on CysLTs production in a well-established mouse infection model. Furthermore, we assessed the effect of anti-inflammatory agents (a leukotriene receptor antagonist, MK-571, and dexamethasone) on the functional and immune changes induced by RSV infection. Six to 8-wk-old BALB/c mice were infected with human RSV (strain A2). Measurements of airway function were performed using whole body plethysmography. Lung inflammation was assessed by cell counts, measurement of cytokines and CysLTs in bronchoalveolar lavage fluid (BALF) in the absence and presence of treatment with MK-571 or dexamethasone. RSV infection produced a marked increase in CysLTs in the BALF and lung tissue, recruitment of neutrophils and lymphocytes into the airways, increased IFN-gamma levels and airway hyperresponsiveness (AHR). Treatment with MK-571 decreased RSV-induced AHR without affecting the cellular and inflammatory responses to RSV. Dexamethasone decreased AHR and markedly reduced the recruitment of inflammatory cells and production of IFN-gamma. Our findings suggest CysLTs play an important role in the pathogenesis of RSV-induced airway dysfunction. Treatment with MK-571 decreases RSV-induced AHR but does not appear to alter the lung inflammatory responses to RSV. In contrast, dexamethasone decreases RSV-induced AHR but interferes with recruitment of inflammatory cells, resulting in decreased Th1 cytokines (a potentially Th2-prone environment) in this model. These studies support recent reports on the beneficial effects of CysLT receptor antagonist in human trials and provide a model for investigating the role of CysLTs in RSV bronchiolitis.     

Respiratory syncytial virus infection reversed anti-asthma effect of neonatal Bacillus Calmette-Guerin vaccination in BALB/c mice

Bacillus Calmette-Guerin (BCG) vaccination can protect animals from asthma, but the effect of BCG on childhood asthma prevention is controversial in humans. To verify the hypothesis that the BCG anti-asthma effect in childhood might be reversed by a respiratory virus infection, newborn BALB/c mice were divided into five groups. Control and ovalbumin (OVA) groups were mock vaccinated and mock infected. The BCG/OVA group was BCG vaccinated and mock infected. The respiratory syncytial virus (RSV)/OVA group was mock vaccinated and RSV infected. The BCG/RSV/OVA group was BCG vaccinated and RSV infected. Except for the control group, all groups underwent OVA sensitization and challenge. Airway hyperresponsiveness (AHR) was measured after challenge and cells in bronchoalveolar lavage fluid (BALF) were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and inflammatory characteristics of lung sections were scored. Mice with neonatal BCG vaccination (BCG/OVA group) were significantly protected from BALF eosinophilia, AHR to methacholine, peribronchiolitis, alveolitis, and peribronchial eosinophilia in comparison with the OVA, RSV/OVA, and BCG/RSV/OVA groups. AHR in the OVA group was greater than in the BCG/OVA group but lower than in the RSV/OVA and BCG/RSV/OVA groups. No significant differences in BALF eosinophilia, AHR, and lung inflammation were found between the RSV/OVA and BCG/RSV/OVA groups. The impact of BCG vaccination on anti-asthma in mice was not dependent on interferon-gamma, IL-4, and IL-10 levels. The results suggested that RSV infection can reverse the anti-asthma effect of neonatal BCG vaccination in BALB/c mice.     

耐受性树突状细胞治疗卵清蛋白致敏小鼠的实验研究

目的 将重组AdCTLA4Ig/Adα4β7诱导的耐受性树突细胞(DC)回输于卵清蛋白(OVA)致敏小鼠体内,观察其对食物过敏小鼠的治疗效果,为临床治疗儿童食物过敏提供新的思路.方法 建立BALB/c小鼠食物过敏模型.BALB/c雌鼠24只,分为3组:肠道激发4 h后回输耐受性DC为治疗组,回输生理盐水为阴性对照组和OVA过敏小鼠为阳性对照组.ELISA测定各组血清中OVA特异性IgE水平;HE染色观察空肠形态;免疫组化法检测小鼠小肠组织中白介素10(IL-10);免疫荧光法检测转化生长因子β(TGF-β)表达水平.结果 ELISA法检测小鼠血清中OVA特异性IgE水平阳性对照组为(0.25±0.05)、阴性对照组为(0.14±0.04)、治疗组为(0.17±0.03).治疗组较阴性对照组小鼠差异无统计学意义(P＞0.05),而较阳性对照组过敏小鼠明显降低,差异有统计学意义(P＜0.05).HE染色可见治疗组小肠绒毛中上皮细胞局灶性坏死、脱落,固有层炎症细胞浸润现象明显改善;IL-10表达增高.各组TGF-β表达无差异.结论 AdCTLA4Ig/Adα4β7修饰的耐受性DC对卵清蛋白过敏小鼠具有治疗作用,可以减轻肠道炎症反应,促进免疫耐受形成.     

金银花水提物对卵清蛋白致敏小鼠的免疫调控作用

目的研究中药金银花水提物（以下简称金银花）对卵清蛋白（OVA）致敏小鼠的免疫调控作用,探索中药治疗食物过敏的可行性。方法取BALB／c小鼠40只,均分为5组,每组8只,按照肠道激发后灌服不同浓度的金银花水提物分为100mg／100ml（H）、50mg／100ml（M）、25mg／100ml（L）浓度组,激发后不给药组（Ch）,以及正常生理盐水（NS）对照组。取空肠行HE及甲苯胺蓝染色;组织荧光法测定小肠组胺含量;ELISA法测定外周淋巴组织单个核细胞（PLNMC）培养上清液中白细胞介素4（IL-4）、γ干扰素（IFN-γ）及血清OVA特异性IgE（OVA-sIgE）水平;RT-PCR测定PLNMC中IL-12p40 mRNA表达;足垫肿胀实验检测迟发型超敏反应。结果H、M组浓度的金银花水提物可缓解过敏小鼠小肠绒毛炎症,减轻肥大细胞聚集和脱颗粒,提高固有层完整肥大细胞比率,减轻过敏小鼠肠道组胺释放,降低过敏小鼠体内IL-4、OVA-sIgE水平及IL-4／IFN-γ比值,抑制PLNMC中IL-12 mRNA表达;三种浓度金银花可缓解OVA介导的小鼠足垫迟发性超敏反应（DTH）。结论证实金银花水提物可参与OVA致敏小鼠的免疫调控,对缓解OVA介导的小鼠IgE及细胞介导的变态反应有利,用于食物过敏治疗具有潜在研究价值。     

Immune responses in rhesus rotavirus-challenged BALB/c mice treated with bifidobacteria and prebiotic supplements

Bifidobacterium species (B. bifidum and B. infantis), with or without prebiotic compounds (arabino-galactan, short-chain fructo-oligosaccharide, iso-malto-dextrins), were orally fed to Balb/c pups (n = 192) to evaluate their potential synergistic effects on modulating the course of rhesus rotavirus (RRV) infection, as well as their ability to mediate the associated mucosal and humoral immune responses. Rotavirus-specific IgA and IgG in serum, rotavirus antigen, and specific IgA in feces were measured by ELISA. Mucosal total IgA and IgG levels were determined in Peyer's patches by flow cytometry. Significantly delayed onset (p = 0.001) and early resolution (p < 0.001) of diarrhea were observed in bifidobacteria-treated, RRV-infected mice compared with RRV-infected control mice. Supplementation with prebiotic compounds did not shorten the clinical diarrhea course more than that observed with bifidobacteria treatment alone. Rotavirus-specific IgA in feces was 16-fold elevated on d 5 postinfection in bifidobacteria-treated, RRV-infected mice compared with the RRV-infected alone group. In addition, the level of rotavirus-specific IgA in serum was four-fold higher in bifidobacteria-treated, RRV-infected litters versus mice challenged with RRV alone on 28 and 42 d postinfection. No enhancement of the immune response was found in RRV-infected mice that were treated with both bifidobacteria and prebiotic compounds over those treated with bifidobacteria only. The findings suggest that bifidobacteria may act as an adjuvant by modulating early mucosal and strong humoral rotavirus-specific immune responses, and mitigate severity of rotavirus-induced diarrhea.     

EL4T淋巴细胞白血病/淋巴瘤小鼠急性移植物抗宿主病动物模型的建立

目的 建立EL4T淋巴细胞自血病/淋巴瘤小鼠急性移植物抗宿主病(acute graft-versus-host disease,aGVHD)的动物模型,为同种异基因骨髓移植(allogeneic bone marrow transplantation,Allo-BMT)中防治aGVHD和保留移植物抗白血病(graft-versus-leukemia,GVL)研究提供理想的模型.方法 以雄性BALB/c(H-2Kd)小鼠为供鼠,雌性C57BL/6(H-2Kb)小鼠为受鼠,受鼠致死性全身照射(9.5 Gy)预处理后,进行Allo-BMT.用随机数字法将受者分为3组,每组17只.(1)单纯照射组:经受着尾静脉输注RPMI 1640培养液(0.2mL/只);(2) aGVHD组:每经尾静脉输注供鼠骨髓细胞(5×106个/只)+脾细胞(5×106个/只);(3) EL4T淋巴细胞白血病/淋巴瘤aGVHD组:经尾静脉输注供鼠骨髓细胞(5×106个/只)+脾细胞(5×106个/只)+ EL4细胞(500个/只).观察受鼠的一般情况、生存期,记录GVHD和白血病发生情况,行组织病理学、免疫组织化学和嵌合体检查.结果 单纯照射组小鼠平均生存时间为(10.10±0.43)d,aGVHD组小鼠平均生存时间(28.12±5.01)d,EL4T淋巴细胞白血病/淋巴瘤aGVHD组小鼠平均生存时间为(31.05 ±5.48)d.aGVHD组和EL4T淋巴细胞白血病/淋巴瘤aGVHD组小鼠平均生存时间均显著长于单纯照射组(P均＜0.01),aGVHD组与EL4T淋巴细胞白血病/淋巴瘤aGVHD组比较差异无统计学意义(P＞0.05).aGVHD组和EL4T淋巴细胞白血病/淋巴瘤aGVHD组小鼠皮肤、肝脏和小肠病理切片存在GVHD病理改变,EL4T淋巴细胞白血病/淋巴瘤aGVHD组小鼠肝脏、脾脏组织病理切片均存在白血病细胞浸润表现.结论 EL4T淋巴细胞白血病/淋巴瘤小鼠aGVHD的动物模型是可靠的,可作为理想的aGVHD动物模型.     

黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠肠道炎症因子和抗炎因子的影响

目的 观察黄褐毛忍冬总皂苷(Ful)对卵清蛋白(OVA)致敏小鼠肠道炎症因子和抗炎因子的影响.方法 24只雌性BALB/c小鼠随机取16只,采用卵清蛋白致敏和激发构建食物过敏模型,均分为2组,即食物过敏组(FA组)和Ful干预组(Ful组).Ful组小鼠自造模第20天起每日皮下注射Ful 200 mg/kg,共22 d.另8只小鼠作为正常对照组(NS组).采用逆转录-聚合酶链反应(RT-PCR)法检测小鼠空肠组织中转化生长因子β1(TGF-β1)、白细胞介素6(IL-6)、白细胞介素17A(IL-17A)、叉头蛋白3-T细胞转录因子(Foxp3)mRNA表达;免疫组织化学法检测小鼠空肠组织中TGF-β1、IL-6、IL-17A蛋白表达;检测空肠中髓过氧化物酶(MPO)活性代表中性粒细胞活化水平.结果 FA组小鼠空肠组织中TGF-β1、IL-6、IL-17A的mRNA[(0.370±0.013)、(0.475±0.015)]和TGF-β1、IL-6、IL-17A蛋白表达水平[(53 075.70±20 727.06)、(256 881.66±36 561.79)、(435 064.25±69 911.48)]均增高,Foxp3 mRNA(0.231±0.014).经黄褐毛忍冬总皂苷干预后,小鼠空肠组织TGF-β1表达未下降,但IL-6、IL-17A的mRNA[(0.196±0.005)、(0.204±0.008)]和蛋白表达水平[(114 040.30±20 295.25)、(218 200.74±30 077.69)]均明显降低,而Foxp3 mRNA(0.578±0.021)表达明显增高.空肠组织中MPO水平各组间比较差异无统计学意义(P＞0.05)..结论 食物过敏发生时肠道内存在以IL-6、IL-17A表达增高为主的炎症反应.Ful可有效降低OVA致敏小鼠肠道炎症因子IL-6、IL-17A的过度表达,显著增强调节性T细胞特异性转录因子Foxp3的表达,从改善OVA诱导的肠道炎症.